The cluster of differentiation, also called cluster of designation (CD) is a nomenclature system used for the identification and investigation of cell surface molecules, either receptors or ligands used for immunophenotyping of cells. T cell receptors (TCR), which reside on the cell surface of T cells, can combine with CD markers forming a complex1,2. This complex can classify the type of T cell and its stage in the cell development process. CD3 is a multimeric protein complex that contains one γ, one δ, two ε and two ζ chains, which associate with TCR to form TCR complex2. In early stages of T-cell development, CD3 is expressed in the cytoplasm and as the T cell matures, it migrates into the cell membrane1,2. CD3 is expressed in all T-cells during all stages of their development making it a specific marker for identification of T-cells1,2.
CD16 is a member of the Fc receptor family which contributes to the protective functions of the immune system and has two subtypes FcγRIIIa (CD16a) and FcγRIIIb (CD16b). Fc receptors are designed to bind antibodies that are attached to foreign cell types4. CD16 is present on the surface of natural killer cells (NK), neutrophils, macrophages, monocytes, and in rare cases activated T cells4. CD16 on NK cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC)3,4,5. Once the Fc receptor on an NK cell recognizes foreign IgG antibody, the NK cells releases cytokines that enter the foreign cell and promotes cell death by apoptosis5.
GA201, which is a glycoengineered anti-EGFR human monoclonal antibody, enhances ADCC6. GA201 is an IgG1 molecule whose Fc region is glycosylated to increase its binding affinity for FcγRIIIa (CD16a) on immune effector cells. Since GA201 was engineered for high ADCC effectiveness, it has demonstrated increased affinity for low and high CD16 expressed on NK cells, macrophages, and T cell sub populations6. This allows CD16 to be used as indicator of the level of GA201 activity in patients with tumors6. CD3 has also been suggested to be linked in GA201 activity through triggering of adaptive immunity. This indicates that detecting CD3 and CD16 together can be used to predict a cancer's likely response to GA2013.
Unfortunately reliable antibodies from different species for CD3 and CD16 are not available. This leads to undesirable cross-reactivity of secondary antibodies used to detect the CD3- and CD16-primary antibodies in a sample. Thus, methods are needed that permit detection of proteins using primary antibodies from the same species (e.g., mouse CD3 and mouse CD16 antibodies, or rabbit CD3 and rabbit CD16 antibodies).